Additionally, a negative control without primary antibody staining is shown
Additionally, a negative control without primary antibody staining is shown. 7-positive lymphocyte migration and retention in the inflamed gut mucosa, but the precise mechanisms by which this inhibition happens are not fully understood. Methods: Cellular effects of etrolizumab or etrolizumab surrogate antibody (etrolizumab-s) were investigated in cell tradition models and analyzed by circulation cytometry, fluorescence microscopy, ImageStream?, stimulated emission depletion (STED) microscopy and practical dynamic adhesion assays. Moreover, effects on 47 integrin were compared with the pharmacodynamically related antibody vedolizumab. Results: As shown by several different methods, etrolizumab and etrolizumab-s treatment led to internalization of 7 integrin. This resulted in impaired dynamic adhesion to MAdCAM-1. Internalized 7 integrin localized in endosomes and re-expression of 7 was dependent on protein synthesis. etrolizumab treatment did not lead to cellular activation or cytokine secretion and did not induce cytotoxicity. Internalization of 47 integrin was improved with etrolizumab compared with vedolizumab. Conversation: Our data suggest that etrolizumab does not elicit secondary effector functions within the solitary cell level. Integrin internalization may be an important mechanism of action of etrolizumab, which might clarify some but not all immunological effects observed with etrolizumab. = 53) and UC (= 44) following prior informed written consent in the Outpatient Division of the Medical Medical center Glucagon receptor antagonists-1 1 of the University or college Hospital Erlangen. Control blood was from healthy donors (= 27). Clinical data of blood donors are summarized in Table ?Table1.1. Blood collection was authorized by the Ethics committee of the Friedrich-Alexander University or college Erlangen-Nuremberg. For some experiments, peripheral blood samples were collected from an anonymous internal Genentech blood donor system of healthy volunteers. Table 1 Patient characteristics. Adhesion Assay Peripheral blood mononuclear cells were cultured for 24 h at 37C in the presence or absence of etrolizumab-s. Next, cells were labeled with carboxyfluorescein succinimidyl ester (CFSE; Existence Systems). Suspensions of 1 1.5 million cells/mL in adhesion buffer (pH 7.4, 150 mM NaCl, 10 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2) were prepared and etrolizumab-s was added or not to aliquots of so far untreated cells. Capillaries for dynamic adhesion assays were prepared as previously explained (Binder et al., 2018). In brief, miniature borosilicate capillaries (Vitrocom) were coated with 5 g/mL rhMAdCAM-1-Fc-chimera (R&D Systems) in 150 mM NaCl with 10 mM HEPES for 1 h at 37C. Next, unspecific binding sites were clogged with 5% bovine serum albumine (BSA) in phosphate buffered saline (PBS) for 1 h at 37C. Perfusion was performed having a peristaltic pump (Shenchen LabV3) at a circulation rate of Glucagon receptor antagonists-1 10 L/min. Dynamic adhesion was analyzed with time-lapse confocal microscopy (Leica SP8) over 3 min Glucagon receptor antagonists-1 and analyzes with ImageJ (NIH) as previously explained (Binder et al., 2018). Immunofluorescence Peripheral blood mononuclear cells were treated with AF647-labeled etrolizumab-s for 24 h at 37 or 4C. In some experiments, cells were permeabilized with 0.1 % Triton X (Roth) after etrolizumab-s incubation and additionally stained with Light-1 (H4A3, AF488, Biolegend) or EEA (5632C2, AF488, Novus Bio) to visualize lysosomes and endosomes, respectively. Subsequently, cells were counterstained with Hoechst dye, suspended in Mowiol (Roth) and covered on microscopy slides. Analyses were performed with fluorescence microscopy (Leica DM6000B). Surface and intracellular fluorescence signals were quantified with ImageJ (NIH) by determining the mean fluorescence intensity (MFI) of regions of interest defined around or in projection to the nuclei, respectively. STED-Microscopy To increase the number of 7 integrin-expressing cells, PBMCs were stimulated with anti-CD3 (OKT3, eBioscience) and anti-CD28 antibodies (Become0248, inVivoMab) and additionally treated with 20 ng/mL TGF- for 72 h as previously explained (Zundler et al., 2017c). Subsequently, such cells were treated having a mouse anti-human 7 antibody (473207, R&D systems) or with or without etrolizumab-s at 37 or 4C for 24 h. Where indicated, cells treated at 37C were additionally permeabilized with 0.1% Triton X. Then, secondary staining was performed with goat Rabbit Polyclonal to GPR37 anti-mouse antibodies and goat anti-rat antibodies labeled with the STED microscopy dye Celebrity 580 (excitation: 594 nm pulsed laser, emission: 605C625 nm) or Celebrity 635P, respectively (both Abberior, excitation: 640 nm pulsed laser, emission: 650C720 nm). Cell suspensions in Mowiol were covered on microscopy slides and analyzed having a STED microscope (Abberior 3D STED 2-Channel Super Resolution Microscope) equipped with a 100 Oil immersion lens (NA: 1.44). Stimulated emission depletion was performed at 775 nm having a pulsed laser. The Glucagon receptor antagonists-1 power of the STED laser was arranged to 625 mW. Antibody-Dependent Cytotoxicity (ADCC) Assay With PBMCs Antibody-dependent cytotoxicity.